Pyridoxine and pyridoxal analogues: new uses

ABSTRACT

The invention provides pyridoxal and pyridoxine analogues, pharmaceutical compositions containing pyridoxine and pyridoxal analogues, and methods of administering pharmaceutical compositions containing a therapeutically effective amount of at least one of these analogues. In accordance with the present invention, the pyridoxal and pyridoxine analogues can be used in the treatment or prevention of of heparin induced thrombocytopenia (HIT), stroke, and ischemia, and in the treatment of symptoms thereof. The the pyridoxal and pyridoxine analogues can be used in neuroprotection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/411, 552, filed Apr. 10, 2003, which is a continuation-in-part of U.S. application Ser. No. 10/147,263, now U.S. Pat. No. 6,548,519, filed on May 15, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/900,718, now U.S. Pat. No. 6,417,204, filed on Jul. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/216,907, filed on Jul. 7, 2000.

FIELD OF THE INVENTION

The present invention relates to pyridoxine and pyridoxal analogue compounds, pharmaceutical compositions containing the pyridoxine and pyridoxal analogue compounds, and methods of treatment using a therapeutically effective amount of the pyridoxine and pyridoxal analogue compounds. The pyridoxine or pyridoxal analogues can be used in the treatment of undesired stroke, ischemia, and heparin induced thrombocytopenia (HIT), or related diseases, and symptoms thereof, as well as providing neuroprotection.

BACKGROUND OF THE INVENTION

Pyridoxal-5′-phosphate (PLP), an end product of vitamin B₆ metabolism, plays a vital role in mammalian health. Vitamin B₆ typically refers to pyridoxine, which is chemically known as 2-methyl-3-hydroxy-4,5-di(hydroxymethyl)pyridine and is represented by formula I:

Yet two additional compounds, pyridoxal of formula II

and pyridoxamine of formula III

are also referred to as vitamin B₆. All three compounds serve as precursors to pyridoxal-5′-phosphate (PLP), which is chemically known as 3-hydroxy-2-methyl-5-[(phosphonooxy) methyl]-4-pyridine-carboxaldehyde and is represented by formula IV:

PLP is the biologically active form of vitamin B₆ inside cells and in blood plasma. Mammals cannot synthesize PLP de novo and must rely on dietary sources of the precursors pyridoxine, pyridoxal, and pyridoxamine, which are metabolized to PLP. For instance, mammals produce PLP by phosphorylating pyridoxine by action of pyridoxal kinase and then oxidizing the phosphorylated product.

PLP is a regulator of biological processes and a cofactor in more than one hundred enzymatic reactions. It has been shown to be an antagonist of a purinergic receptor, thereby affecting ATP binding; it has been implicated in modulation of platelet aggregation; it is an inhibitor of certain phosphatase enzymes; and it has been implicated in the control of gene transcription. In previous patents (U.S. Pat. No. 6,051,587 and U.S. Pat. No. 6,043,259) the role of pyridoxal-5′-phosphate, and its precursors pyridoxal and pyridoxine (vitamin B₆), in mediating cardiovascular health and in treating cardiovascular related diseases is disclosed. PLP is also a coenzyme in certain enzyme-catalyzed processes, for example, in glycogenolysis at the glycogen phosphorylase level, in the malate asparatate shuttle involving glycolysis and glycogenolysis at the transamination level, and in homocysteine metabolism.

There is a need to identify and administer drugs that can mimic one or more of the known biological actions of vitamin B-6 congeners but that are more potent than the vitamin B-6 congeners in their specific mode of action.

SUMMARY OF THE INVENTION

The present invention provides for pyridoxine and pyridoxal analogues, pharmaceutical compositions containing the pyridoxine and pyridoxal analogues, and methods for treatment based on administration of therapeutically effective amounts of the pyridoxine and pyridoxal analogues. Compounds and compositions of the invention can be used for the treatment of cardiovascular or related diseases and symptoms thereof.

The invention provides pyridoxine and pyridoxal analogues of Formula V:

or a pharmaceutically acceptable acid addition salt addition salt thereof, wherein:

-   -   R₅ is CH₂OH or CHO;     -   R₁ is     -   n is an integer of 1 to 5;     -   R₂, R₃, and R₄ are each independently         -   hydrogen;         -   alkyl;         -   aryl or biaryl,             -   wherein the aryl or biaryl can be substituted with a                 cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or                 alkanoyloxy;         -   amino;         -   acylamino;         -   anilino,             -   wherein the aniline ring can be substituted with a                 cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or                 alkanoyloxy;         -   nitro; or         -   guanidino.

In another aspect, the invention is directed to a pharmaceutical composition that includes a pharmaceutically acceptable carrier in combination with a therapeutically effective amount of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V.

In another aspect, the invention is directed to a method of treating cardiovascular or related diseases and symptoms thereof. The method includes administering to a mammal a therapeutically effective amount of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V in a unit dose form. The method can further include concurrent administration of another therapeutic agent.

In another aspect, the invention is directed to a method of reducing platelet aggregation and the symptoms there of a mammalian patient. The method includes administering to the mammalian patient a therapeutically effective amount of a compound of Formula V. In some instances the patient may suffer from heparin-induced thrombocytopenia (HIT).

In another aspect, the invention is directed to a method of reducing or preventing heparin-induced thrombus formation in a mammalian patient suffering from HIT. The method includes administering to the mammalian patient a therapeutically effective amount of a compound of Formula V.

In another aspect, the invention is directed to a method of reducing or preventing brain damage in a mammalian patient suffering from stroke or ischemia. The method includes administering to the mammalian patient a therapeutically effective amount of a compound of Formula V.

In another aspect, the invention is directed to a method of reducing brain damage in a mammalian patient by neuroprotection. The method includes administering to the mammalian patient a therapeutically effective amount of a compound of Formula V.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides pyridoxal and pyridoxine analogues and pharmaceutical compositions containing these pyridoxine and pyridoxal analogues. The pyridoxine and pyridoxal analogues can be used in the treatment of cardiovascular or related diseases and symptoms thereof.

Cardiovascular or related diseases include, for example, cerebral ischemia, cerebral hemorrhage, ischemic stroke, hemorrhagic stroke, hypertension, myocardial infarction, ischemia reperfusion injury, myocardial ischemia, congestive heart failure, blood coagulation disorders, cardiac hypertrophy, and platelet aggregation. Cardiovascular or related diseases also includes diseases that arises from thrombotic and prothrombotic states in which the coagulation cascade is activated such as, for example, deep vein thrombosis, disseminated intravascular coagulopathy, and pulmonary embolism.

Heart failure is a pathophysiological condition in which the heart is unable to pump blood at a rate commensurate with the requirement of the metabolizing tissues or can do so only from an elevated filling pressure (increased load). Thus, the heart has a diminished ability to keep up with its workload. Over time, this condition leads to excess fluid accumulation, such as peripheral edema, and is referred to as congestive heart failure.

When an excessive pressure or volume load is imposed on a ventricle, myocardial hypertrophy (i.e., enlargement of the heart muscle) develops as a compensatory mechanism. Hypertrophy permits the ventricle to sustain an increased load because the heart muscle can contract with greater force. However, a ventricle subjected to an abnormally elevated load for a prolonged period eventually fails to sustain an increased load despite the presence of ventricular hypertrophy, and pump failure can ultimately occur.

Heart failure can arise from any disease that affects the heart and interferes with circulation. For example, a disease that increases the heart muscle's workload, such as hypertension, will eventually weaken the force of the heart's contraction. Hypertension is a condition in which there is an increase in resistance to blood flow through the vascular system. This resistance leads to increases in systolic pressure, diastolic blood pressure, or both. Hypertension places increased tension on the left ventricular myocardium, causing it to stiffen and hypertrophy, and accelerates the development of atherosclerosis in the coronary arteries. The combination of increased demand and lessened supply increases the likelihood of myocardial ischemia leading to myocardial infarction, sudden death, arrhythmias, and congestive heart failure.

Ischemia is a condition in which an organ or a part of the body fails to receive a sufficient blood supply. When an organ is deprived of a blood supply, it is said to be hypoxic. An organ will become hypoxic even when the blood supply temporarily ceases, such as during a surgical procedure or during temporary artery blockage. Ischemia initially leads to a decrease in or loss of contractile activity. When the organ affected is the heart, this condition is known as myocardial ischemia, and myocardial ischemia initially leads to abnormal electrical activity. This can generate an arrhythmia. When myocardial ischemia is of sufficient severity and duration, cell injury can progress to cell death—i.e., myocardial infarction—and subsequently to heart failure, hypertrophy, or congestive heart failure.

Ischemic reperfusion of the organ occurs when blood flow resumes to an organ after temporary cessation. For example, reperfusion of an ischemic myocardium can counter the effects of coronary occlusion, a condition that leads to myocardial ischemia. Ischemic reperfusion to the myocardium can lead to reperfusion arrhythmia or reperfusion injury. The severity of reperfusion injury is affected by numerous factors, such as, for example, duration of ischemia, severity of ischemia, and speed of reperfusion. Conditions observed with ischemia reperfusion injury include neutrophil infiltration, necrosis, and apoptosis.

Heparin-induced thrombocytopenia (“HIT”) is an immune-mediated syndrome. HIT is caused by antibodies (HIT-Ig) which typically recognize a complex of platelet factor 4 and heparin. The formation of the antibody/antigen complex activates platelets and endothelial cells and generates procoagulent microparticles. Clinical features of HIT can include thrombocytopenia, thrombosis, and heparin-induced skin lesions. (Warkentin, Ann. Rev. Med., 50: 129-147 (1999)).

HIT is characterized by an abrupt reduction in platelet count following treatment of the patient with heparin. It can also cause the formation of clots on subsequent treatment with heparin. Previous attempts to identify compounds effective in preventing this subsequent immune-induced (heparin-induced) clotting have not proven entirely satisfactory.

The exact dose and route of administration of the compound of Formula V will vary based on the patient's size, condition, gender, and medical history. In some instances it will be desirable to administer a compound of Formula V to achieve a plasma concentration of between about 1 μM and 1000 μM. In some instances a plasma concentration of between about 300 μM and 700 μM will be desirable. In some instances a plasma concentration of between about 450 μM and 550 μM will be desirable.

In some instances, it may be desirable to administer compound V intravenously at a dose of between about 2 to 100 mg/kg body weight. In some instances this dose will be administered between one and four times daily to maintain a desired plasma concentration of the compound.

The compounds of the invention may be administered to induce a plasma level of said compound of between about 100 nM and 10,000 μM. They may be administered to a therapeutically effective dose between about 0.1 to 100 mg/kg body weight per day. The may be administered to a dosage between 10 mg/kg and 100 mg/kg body weight. They may be administered to a dosage is between 20 mg/kg and 80 mg/kg body weight.

The compounds of the invention may be administered intravenously, orally, sublingually, intraperitoneally, transdermally, intramuscularly or subcutaneously. In a preferred embodiment, they may be administered intravenously. In a further preferred embodiment, they may be administered orally.

Pyridoxal and Pyridoxine Analogue Compounds

The invention provides pyridoxal and pyridoxine analogue compounds of Formula V:

or pharmaceutically acceptable acid addition salts thereof, wherein:

-   -   R₅ is CH₂OH or CHO;     -   R₁ is     -   n is an integer of 1 to 5;     -   R₂, R₃, and R₄ are each independently         -   hydrogen;         -   alkyl;         -   aryl or biaryl;             -   wherein the aryl or biaryl can be substituted with a                 cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or                 alkanoyloxy;         -   amino;         -   acylamino;         -   anilino,             -   wherein the aniline ring can be substituted with a                 cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or                 alkanoyloxy;         -   nitro; or         -   guanidino.

As used herein, the term “alkyl” refers to a straight or branched saturated aliphatic hydrocarbon chain having 1 to 8 carbon atoms, such as, for example, methyl, ethyl, propyl, isopropyl(1-methylethyl), butyl, tert-butyl(1,1-dimethylethyl), and the like. The alkyl chain can be interrupted by a heteroatom, such as, for example, a nitrogen, sulfur, or oxygen atom, forming an alkylaminoalkyl, alkylthioalkyl, or alkoxyalkyl. Examples of alkyl chain interrupted by a heteroatoms include methylaminoethyl, ethylthiopropyl, methoxymethyl, and the like. The alkyl can be substituted at the terminal carbon by groups such as hydroxy, alkoxy, alkanoyloxy, alkoxycarbonyl, or carboxy.

The term “alkoxy” refers to an alkyl group joined to an oxygen atom. In some embodiments, the alkoxy has 1 to 4 carbon atoms in a straight or branched chain, such as, for example, methoxy, ethoxy, propoxy, isopropoxy(1-methylethoxy), butoxy, tert-butoxy (1,1-dimethylethoxy), and the like.

As used herein, the term “alkanoyloxy” refers to a group of formula

Examples of an alkanoyloxy include methanoyloxy, ethanoyloxy, propanoyloxy, and the like.

The term “halo” refers to a bromo, chloro, or fluoro group. In some embodiments, the halo is fluoro.

Pharmaceutically acceptable acid addition salts of the compounds of Formula V include salts derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, hydrofluoric, phosphorous, and the like, as well as the salts derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Also contemplated are salts of amino acids such as arginate and the like and gluconate, galacturonate, n-methyl glutamine, etc. (see, e.g., Berge et al., J. Pharmaceutical Science, 66: 1-19 (1977).

The acid addition salts of the basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.

In one embodiment of Formula V, R₁ is

Preferably, R₂ is hydrogen, alkyl, or amino.

In another embodiment of Formula V, R₁ is

Preferably, R₂ and R₃ are each independently hydrogen, alkyl, amino, or nitro.

In another embodiment of Formula V, R₁ is

Preferably, R₂, R₃, and R₄ are independently hydrogen, alkyl, or amino. In a particularly preferred embodiment, R₂ is hydrogen, R₃ is methyl, and R₄ is hydrogen.

In another embodiment of Formula V, R₁ is

In still another embodiment of Formula V, R₁ is

In a particularly preferred embodiment, R₁ is

Methods of Preparing Pyridoxal and Pyridoxine Analogue Compounds

Another aspect of the invention provides a method for preparing the pyridoxine and pyridoxal analogues. The compounds of the invention can be prepared from a compound of Formula VI, VII, VIII, IX, or X:

R₆=(CH₂)_(p)OH where p=1 to 5  VI R₆=(CH₂)_(q)Br where q=1 to 5  VII R₆=(CH₂)_(r)CHO where r=0 to 4  VIII R₆=(CH₂)_(n)N₃ where s=1 to 5  IX R₆=(CH₂)_(t)NH₂ where t=1 to 5

A compound of Formula VI, VII, VIII, IX, or X can be used to form the compounds of Formula V through a series of chemical reactions to produce pyridoxine analogues. The pyridoxine analogues can be subsequently oxidized to produce the corresponding pyridoxal analogues.

In some embodiments of the invention, the pyridoxine and pyridoxal analogues are formed by reacting a bromide compound of Formula VII with a substituted or unsubstituted tetrazole, a substituted or unsubstituted triazole, or a substituted or unsubstituted imidazole. The tetrazole, triazole, or imidazole can be substituted with an aryl, biaryl, amino, acylamino, anilino, or guanidine. An aryl or biaryl can be further substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy group. An aniline can be further substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy group.

For example, as shown in Scheme 1, a bromide compound of Formula VII (q=1) can be reacted with 1H-tetrazole to produce derivative XI. The derivative XI is then treated with acetic acid to produce 5-tetrazolepyridoxine XII. Oxidation of 5-tetrazolepyridoxine XII in the presence of a catalyst such as manganese dioxide can be use to produce the corresponding pyridoxal XIII.

In other embodiments of the invention, an aldehyde of Formula VIII is formed by reacting an alcohol of Formula VI with a suitable oxidizing agent such as manganese doxide. The pyridoxine and pyridoxal analogues of Formula V are formed by reacting an aldehyde of Formula VIII with a substituted or unsubstituted triazole, a substituted or unsubstituted imidazole, or a substituted or unsubstituted alinine. The triazole, or imidazole can be substituted with an aryl, biaryl, amino, acylamino, anilino, or guanidine. An aryl or biaryl can be further substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy group. An aniline can be further substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy group.

For example, an aldehyde of Formula VIII (r=0) can react with 2-methylimidazoline to form the protected imidazoline derivative XXVIII according to Scheme 2. The protected imidazoline derivative XXVII can be hydrolyzed to the imidazoline XXIX.

In another example, a compound of the invention can be prepared by reacting an aldehyde of Formula VIII (r=0) with 4-cyano aniline as shown in Scheme 3 to form a Schiff base. The Schiff base XXXII is reacted with a strong reducing agent such as, for example, sodium borohydride. The resulting amine XXXIII can react with ethanol in the presence of dry hydrogen chloride gas to form a compound of Formula XXXIV. The compound of Formula XXXIV can be treated with 2 M NH₃ in MeOH in a pressurized vessel to form the compound of Formula XXXV.

In another embodiment of preparing the compounds of Formula V, an amine of Formula X can react with a substituted guanidine.

For example, as shown in Scheme 4, an amine compound of Formula X (s=1) can react with a protected guanidine compound to give the guanidine derivative XXIII. The protection groups can be removed with trifluoroacetic acid to form a compound of Formula XXV.

In another embodiment of preparing the compounds of Formula V, an azide of Formula IX is the precursor. The isopropylidene group is hydrolyzed initially. The hydroxy groups formed from the hydrolysis reaction are then protected by reacting with a reagent such as, for example, tert-butyldimethylsilyl chloride. The azide group can be hydrogenated to an amine group. The resulting amine compound can react with an aromatic group containing a substituted or unsubstituted aryl or biaryl isocyanate or a substituted thioisocyanate. The aryl or biaryl can be substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy. The protection groups added after the hydrolysis reaction can be removed to form a pyridoxine compound.

For example, as shown in Scheme 5, an arylurea and arylthiourea substituted compound of the invention can be prepared using an azide compound of Formula IX (s=1). The isopropylidene group can be hydrolyzed by treatment with acetic acid to form compound of Formula XLI. The unprotected hydroxyl groups are reacted with tert-butyldimethylsilyl chloride to form XLII. The azide group can be hydrogenated with hydrogen in the presence of a catalyst to form the amine XLIII. The amine can react with 4-fluorophenylisocyanate givning compound XLIV. The syntheses of compounds XLVI and XLVII followed the procedure outlined for XLIV and XLV using 4-fluorophenylthioisocyanate in place of 4-fluorophenylisocyanate.

The products of the reactions described herein are isolated by conventional means such as extraction, distillation, chromatography, and the like.

One skilled in the art can recognize other variations in the reaction sequences and in the appropriate reaction conditions from the analogous reactions shown or otherwise known that may be appropriately used in the above-described processes to make the compounds of Formula V herein.

Pharmaceutical Compositions

Although it is possible for a pyridoxine and pyridoxal analogue compound of the invention to be administered alone in a unit dosage form, the compounds are typically administered in admixture as a pharmaceutical composition to provide a unit dosage form. The invention provides pharmaceutical compositions containing at least one pyridoxine or pyridoxal analogues compound of Formula V. A pharmaceutical composition comprises a pharmaceutically acceptable carrier in combination with a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V.

A pharmaceutically acceptable carrier includes, but is not limited to, physiological saline, ringers, phosphate-buffered saline, and other carriers known in the art. Pharmaceutical compositions can also include additives such as, for example, stabilizers, antioxidants, colorants, excipients, binders, thickeners, dispersing agents, readsorpotion enhancers, buffers, surfactants, preservatives, emulsifiers, isotonizing agents, and diluents. Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective.

Methods of preparing pharmaceutical compositions containing a pharmaceutically acceptable carrier in combination with a therapeutic compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V are known to those of skill in the art. All methods can include the step of bringing the compound of the invention in association with the carrier and additives. The formulations generally are prepared by uniformly and intimately bringing the compound of the invention into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired unit dosage forms.

For oral administration as a suspension, the compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. The compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents. As immediate release tablets, the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

For administration by inhalation or aerosol, the compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. The compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

For rectal administration as suppositories, the compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.

Method of Treatment Using Pyridoxal and Pyridoxine Analogue Compounds

In another aspect of the invention, methods are provided for the treatment of cardiovascular or related diseases and symptoms thereof.

As used herein the terms “treatment” and “treating” as used herein include preventing, inhibiting, alleviating, and healing vitamin B₆ cardiovascular or related diseases or symptoms thereof. Treatment can be carried out by administering a therapeutically effective amount of a compound of the invention. A “therapeutically effective amount” as used herein includes a prophylactic amount, for example, an amount effective for preventing or protecting against the above mentioned diseases or symptoms thereof; or an amount effective for alleviating or healing the above mentioned diseases or symptoms thereof.

A physician or veterinarian of ordinary skill readily determines a mammalian subject who is exhibiting symptoms of any one or more of the diseases described above. Regardless of the route of administration selected, a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V can be formulated into pharmaceutically acceptable unit dosage forms by conventional methods known in the pharmaceutical art. An effective but nontoxic quantity of the compound is employed in treatment. The compounds can be administered in enteral unit dosage forms, such as, for example, tablets, sustained-release tablets, enteric coated tablets, capsules, sustained-release capsules, enteric coated capsules, pills, powders, granules, solutions, and the like. They can also be administered parenterally, such as, for example, subcutaneously, intramuscularly, intradermally, intramammarally, intravenously, and by other administrative methods known in the art.

The ordinarily skilled physician or veterinarian will readily determine and prescribe the therapeutically effective amount of the compound to treat the disease for which treatment is administered. In so proceeding, the physician or veterinarian could employ relatively low dosages at first, subsequently increasing the dose until a maximum response is obtained. Typically, the particular disease, the severity of the disease, the compound to be administered, the route of administration, and the characteristics of the mammal to be treated, for example, age, sex, and weight, are considered in determining the effective amount to administer. Administering a therapeutic amount of a compound of the invention for treating cardiovascular or related diseases or symptoms thereof, is in a range of about 0.1-100 mg/kg of a patient's body weight, more preferably in the range of about 0.5-50 mg/kg of a patient's body weight, per daily dose. The compound can be administered for periods of short and long duration. Although some individual situations can warrant to the contrary, short-term administration, for example, 30 days or less, of doses larger than 25 mg/kg of a patient's body weight is preferred to long-term administration. When long-term administration, for example, months or years, is required, the suggested dose usually does not exceed 25 mg/kg of a patient's body weight.

A therapeutically effective amount of a compound of Formula V or a pharmaceutically acceptable addition salt of a compound of Formula V for treating the above-identified diseases or symptoms thereof can be administered prior to, concurrently with, or after the onset of the disease or symptom. A compound of the invention can be administered concurrently. “Concurrent administration” and “concurrently administering” as used herein includes administering a compound of the invention and another therapeutic agent in admixture, such as, for example, in a pharmaceutical composition or in solution, or separately, such as, for example, separate pharmaceutical compositions or solutions administered consecutively, simultaneously, or at different times but not so distant in time such that the compound of the invention and the other therapeutic agent cannot interact and a lower dosage amount of the active ingredient cannot be administered.

In one embodiment of the invention, a method is provided for treating cardiovascular or related diseases comprising administering to a mammal a therapeutically effective amount of a compound of Formula V or a pharmaceutically acceptable addition salt of a compound of Formula V in a unit dosage form. The cardiovascular or related diseases that can be treated include hypertrophy, hypertension, congestive heart failure, heart failure subsequent to myocardial infarction, myocardial ischemia, cerebral ischemia, ischemia reperfusion injury, arrhythmia, myocardial infarction, blood coagulation, or platelet aggregation. Preferably, the cardiovascular disease treated is hypertrophy, congestive heart failure, arrhythmia, or ischemia reperfusion injury.

The compound of the invention can also be administered to treat cardiovascular diseases and other diseases that arise from thrombotic and prothrombotic states in which the coagulation cascade is activated, such as, for example, deep vein thrombosis, disseminated intravascular coagulopathy, Kasabach-Merritt syndrome, pulmonary embolism, myocardial infarction, stroke, thromboembolic complications of surgery, and peripheral arterial occlusion. A compound of the invention may also be useful in the treatment of adult respiratory distress syndrome, septic shock, septicemia, or inflammatory responses, such as edema and acute or chronic atherosclerosis, because thrombin has been shown to activate a large number of cells outside of the coagulation process, such as, for example, neutrophils, fibroblasts, endothelial cells, and smooth muscle cells.

The method for treating cardiovascular or related diseases can further comprise concurrent administration of other therapeutic agents already known to be suitable for treating the above-identified diseases. For example, methods of the invention include concurrently administering a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V in combination with a therapeutic cardiovascular compound to treat hypertrophy, hypertension, congestive heart failure, heart failure subsequent to myocardial infarction, myocardial ischemia, ischemia reperfusion injury, arrhythmia, or myocardial infarction. Preferably, the cardiovascular disease treated is hypertrophy, congestive heart failure, arrhythmia, or ischemia reperfusion injury.

Other therapeutic cardiovascular compounds that can be concurrently administered with a compound or composition of the invention include an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, an antithrombolytic agent, a β-adrenergic receptor antagonist, a vasodilator, a diuretic, an α-adrenergic receptor antagonist, an antioxidant, and a mixture thereof. In one embodiment, a compound of the invention is administered concurrently with PPADS (pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid), also a therapeutic cardiovascular compound, or concurrently with PPADS and another known therapeutic cardiovascular compound as already described.

Preferably the other therapeutic cardiovascular compound, which is concurrently administered with a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V, is an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a diuretic, an α-adrenergic receptor antagonist, or a calcium channel blocker.

Known angiotensin converting enzyme inhibitors include, for example, captopril, enalapril, lisinopril, benazapril, fosinopril, quinapril, ramipril, spirapril, imidapril, and moexipril.

Examples of known angiotensin II receptor antagonists include both angiotensin I receptor subtype antagonists and angiotensin II receptor subtype antagonists. Suitable antiotensin II receptor antagonists include losartan and valsartan.

Suitable calcium channel blockers include, for example, verapamil, diltiazem, nicardipine, nifedipine, amlodipine, felodipine, nimodipine, and bepridil.

Examples of known β-adrenergic receptor antagonists include atenolol, propranolol, timolol, and metoprolol.

Suitable vasodilators include, for example, hydralazine, nitroglycerin, and isosorbide dinitrate.

Suitable diuretics include, for example, furosemide, diuril, amiloride, and hydrodiuril.

Suitable α-adrenergic receptor antagonists include, for example, prazosin, doxazocin, and labetalol.

Suitable antioxidants include vitamin E, vitamin C, and isoflavones.

These other therapeutic cardiovascular compounds are generally used to treat cardiovascular or related diseases as well as symptoms thereof. A skilled physician or veterinarian readily determines a subject who is exhibiting symptoms of any one or more of the diseases described above and makes the determination about which compound is generally suitable for treating specific cardiovascular conditions and symptoms.

For example, myocardial ischemia can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, a angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, an antithrombolytic agent, a β-adrenergic receptor antagonist, a diuretic, an I-adrenergic receptor antagonist, or a mixture thereof.

As another example, congestive heart failure can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, a angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, a vasodilator, a diuretic, or a mixture thereof.

Myocardial infarction can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, a angiotensin converting enzyme inhibitor, a calcium channel blocker, an antithrombolytic agent, a β-adrenergic receptor antagonist, a diuretic, an α-adrenergic receptor antagonist, or a mixture thereof.

Hypertension can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, a calcium channel blocker, a β-adrenergic receptor antagonist, a vasodilator, a diuretic, an α-adrenergic receptor antagonist, or a mixture thereof.

Arrhythmia can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, a calcium channel blocker, a β-adrenergic receptor antagonist, or a mixture thereof.

Blood clots in the arteries can be reduced or removed by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with an antithrombolytic agent. Antithrombolytic agents known in the art include antiplatelet agents, aspirin, and heparin.

Hypertrophy can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, or a mixture thereof.

Ischemia reperfusion injury can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, or a mixture thereof.

Platelet aggregation can be treated by the administration of a compound of Formula V or a pharmaceutically acceptable acid addition salt of a compound of Formula V concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, Danaparoid; Lepirudin; Hirudin; Argatroban; and in some instances, Heparin and/or Warfarin; or a mixture thereof.

This invention is further characterized by the following examples. These examples are not meant to limit the scope of the invention but are provided for exemplary purposes to more fully describe the invention. Variation within the scope of the invention will be apparent to those skilled in the art.

EXAMPLES

Preparation of Starting Materials

Bromide VII (q=1) was prepared by a literature procedure; Imperalli et al, J. Org. Chem., 60, 1891-1894 (1995). Alcohol VI (p=1) was prepared by bubbling HCl gas into a solution of pyridoxine hydrochloride (50 g, 0.24 mol) in acetone (500 mL) at 0-5° C. (ice bath) until the solution became clear. Diethyl ether (ca. 1 L) was added to induce precipitation of the hydrochloride salt which was filtered off. The salt was dissolved in a mixture of methylene chloride (ca. 1 L) and saturated aqueous NaHCO₃ (ca. 500 mL). The layers were separated and the organic layer washed with saturated aqueous NaHCO₃. The combined organic layers were dried (MgSO₄), and evaporated to give 40.5 g (80%) of a colourless solid.

Alcohol VI (p=1) was dissolved in dichloromethane and cooled to 0° C. Small of amounts of triphenylphosphine and N-bromosuccinimide were added alternately over a period of about five minute. The reaction mixture was stirred for about 20 minutes and then concentrated in vacuo. The crude product, bromide VII (q=1) was purified by flash chromatography using a 2:1 mixture of ether and hexanes as the eluent. The product was used immediately.

Aldehyde VIII (r=0) was identified by comparison to data in the literature; Kortynk et al., J. Org. Chem., 29, 574-579 (1964). MnO₂ (Aldrich 21,764-6) (49.9 g, 85%, 487 mmol) was added to a solution of alcohol VI (p=1) (25 g, 119.6 mmol) in toluene (900 ml). The resulting mixture was stirred at 40° C. for 24 hours then filtered through Celite. The mother liquor was evaporated to give a light yellow solid. The solid was recrystallized from hexane:ethyl ether (1:1) to give a light yellow solid. The solid was filtered and washed with hexane:ethyl ether (1:1) to give the pure aldehyde VIII (17.51 g, 71%).

Azide IX (s=1) and amine X (t=1) were prepared from bromide VII (q=1). Bromide VII (q=1) (1.08 g. 4.0 mmol) in anhydrous DMF (20 ml) was treated with sodium azide (260 mg, 4.0 mmol) at room temperature. After one hour stirring at room temperature, the solution was extracted with diethyl ether (5×20 mL). The combined extracts were washed with water (10 mL) and brine (10 mL) followed by drying (MgSO₄). The solvent was evaporated and the crude product was purified by chromatography on silica gel using ethyl ether: hexanes (2:1) as eluent to give the azide IX (s=1) as a colourless liquid (552 mg, 60%). ¹H NMR (CDCl₃, TMS) δ 1.57 (s, 6H), 2.42 (s, 3H), 4.23 (s, 2H), 4.86 (s, 2H), 7.96 (s, 1H).

The purified azide IX (s=1) (100 mg, 0.4 mmol) was dissolved in 95% ethanol and hydrogenated at 1 atm in presence of Lindlar catalyst (50 mg) for one hour. The catalyst was removed by filtration (Celite), and the solvent removed to give the crude amine X (t=1). Purification by chromatography on silica gel using CH₂Cl₂:MeOH (5:1) as eluent gave the product (80 mg, 82%) ¹HNMR (CD₂Cl₂) 1.53 (s, 6H), 2.34 (s, 3H), 3.72 (s, 2H), 4.91 (s, 2H), 5.31 (s, 2H), 7.93 (s, 1H).

All other reagents used in the following examples can be purchased from Aldrich Chemical Company (Milwaukee, Wis. or Allentown, Pa.).

Example 1 Synthesis of Tetrazole Substituted Pyridoxine Analogue of Formula XII

A mixture of tetrazole (94.2 mg, 1.29 mmol) and pulverized anhydrous potassium carbonate (1.5 g) in anhydrous acetonitrile (10 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (350 mg, 1.29 mmol) in anhydrous acetonitrile (3 mL) was then added to the reaction mixture maintaining the reaction temperature for the next 30 minutes. After the completion of the reaction, routine work-up gave the crude product. Purification of the crude mixture on silica gel column gave the desired product XI in appreciable yields.

¹H nmr (CDCl₃, TMS): δ 1:52 (6H, s), 2.44 (3H, s), 4.77 (2H, s), 5.47 (2H, s), 8.07 (1H, s), 8.55 (1H, s, -tetrazole-H).

The purified derivative XI (100 mg, 0.4 mmol) was then taken in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 5-tetrazolepyridoxine XII in good yields.

¹H nmr (CD₃OD, TMS): δ 2.42 (3H, s), 4.96 (2H, s), 5.97 (2H, s), 7.92 (1H, s), 8.69 (1H, s, terazole H).

Example 2 Synthesis of Tetrazole Substituted Pyridoxal Analogue of Formula XIII

The 5-tetrazole pyridoxine XII (100 mg, 0.42 mmol) was dissolved in anhydrous toluene (10 mL). To the solution was added activated manganese dioxide (243 mg, 2.76 mmol), and the reaction mixture heated at 40° C. for 2 hours to ensure complete oxidation. Filtration of the catalyst, followed by evaporation of the solvent gave the crude residue which was easily purified by chromatography on silica gel to give the desired aldehyde XIII in 70% yield.

¹H nmr (CD₂Cl₂, TMS): δ 2.82 (3H, s), 6.00 (1H, s), 6.15 (1H, s), 8.11 (1H, s), 8.57 (1H, s, tetrazole-H), 10.77 (1H, s, aldeydic-H).

Example 3 Synthesis of Tetrazole Substituted Pyridoxine Analogue of Formula XV

A mixture of tetrazole (94.2 mg, 1.29 mmol) and pulverized anhydrous potassium carbonate (1.5 g) in anhydrous acetonitrile (10 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (350 mg, 1.29 mmol) in anhydrous acetonitrile (3 mL) was then added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on silica gel gave the desired product XIV in appreciable yields.

¹H nmr (CDCl₃, TMS): δ 1:53 (6H, s), 2.42 (3H, s), 4.91 (2H, s), 5.66 (2H, s), 8.14 (1H, s), 8.50 (1H, s, -tetrazole-H).

The purified derivative XIV (100 mg, 0.4 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel column gave the 5-tetrazole pyridoxine XV in good yields.

¹H nmr (CD₃OD, TMS): δ 2.43 (3H, s), 4.89 (2H, s), 5.77 (2H, s), 7.91 (1H, s), 9.17 (1H, s, terazole H).

Example 4 Synthesis of Tetrazole Substituted Pyridoxine Analogue of Formula XVII

A mixture of aminotetrazole (110.2 mg, 1.30 mmol) and pulverized anhydrous potassium carbonate (1.5 g) in anhydrous acetonitrile (10 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (360 mg, 1.30 mmol) in anhydrous acetonitrile (3 mL) was added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on a silica gel column gave the desired product XVI in appreciable yields.

¹H nmr (CD₃OD, TMS): δ 1:52 (6H, s), 2.36 (3H, s), 4.96 (2H, s), 5.56 (2H, s), 7.96 (1H, s).

The purified derivative XVI (100 mg, 0.37 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 5-tetrazole pyridoxine XVII in good yields.

¹ Hnmr (CD₃OD, TMS): δ 2.42 (3H,s), 4.94 (2H, s), 5.66 (2H, s), 7.91 (1H, s), 7.87 (1H, s).

Example 5 Synthesis of Triazole Substituted Pyridoxine Analogue of Formula XIXa

A mixture of triazole (136 mg, 2.00 mmol) and pulverized anhydrous potassium carbonate (2.5 g) in anhydrous acetonitrile (20 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (720 mg, 2.00 mmol) in anhydrous acetonitrile (5 mL) was added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on a silica gel column gave the desired product XVIIIa in appreciable yield.

¹H nmr (CDCl₃, TMS): δ 1:53 (6H, s), 2.42 (3H, s), 4.80 (2H, s), 5.24 (2H, s), 7.94 (1H,s,-triazole-H), 7.99 (1H, s, -triazole-H), 8.15 (1H, s).

The purified derivative XVIIIa (100 mg, 0.35 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. Purification by chromatography on silica gel column gave the 5-triazole pyridoxine XIXa in good yield.

¹H nmr (CD₃OD, TMS): δ 2.42 (3H, s), 4.91 (2H, s), 5.50 (2H, s), 7.82 (1H, s, -triazole-H), 7.97 (1H, s, -triazole-H), 8.52 (1H, s).

Example 6 Synthesis of Triazole Substituted Pyridoxal Analogue of Formula XXa

Following Scheme 8,5-triazole pyridoxine XIXa (100 mg, 0.42 mmol) was dissolved in anhydrous toluene (10 mL). To the solution was added activated manganese dioxide (243 mg, 2.76 mmol), and the reaction mixture heated at 40° C. for 2 hours to ensure complete oxidation. Filtration of the catalyst, followed by evaporation of the solvent gave a crude residue that was easily purified by chromatography on silica gel to give the desired aldehyde XXa in 70% yield.

¹H nmr (CD₃OD, TMS): δ 2.70 (3H, s), 6.00 (2H, s), 6.28 (2H, s), 8.41 (1H, s, -triazole-H), 8.85 (1H, s, -triazole-H), 9.85 (1H, s).

Example 7 Synthesis of Triazole Substituted Pyridoxine Analogue of Formula XIXb

Following Scheme 8, a mixture of 3-aminotriazole (142 mg, 2.00 mmol) and pulverized anhydrous potassium carbonate (2.5 g) in anhydrous acetonitrile (20 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (720 mg, 2.00 mmol) in anhydrous acetonitrile (5 mL) was added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on silica gel the desired product XVIIIb in appreciable yields.

¹H nmr (CD₃OD, TMS): δ 1.57 (6H, s), 2.36 (3H, s), 4.84 (2H, s), 4.99 (2H, s), 7.84 (1H, s, -triazole-H), 8.13 (1H, s).

The purified derivative XVIIIb (100 mg, 0.35 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 3-aminotriazole pyridoxine XIXb in good yields.

¹H nmr (DMSO-d₆, TMS): δ 2.34 (3H, s), 4.72 (2H, s), 5.17 (2H, s), 7.79 (1H, s, -triazole-H), 8.03 (1H, s).

Example 8 Synthesis of Triazole Substituted Pyridoxine Analogue of Formula XIXc

Following Scheme 8, a mixture of 5-aminotriazole (142 mg, 2.00 mmol) and pulverized anhydrous potassium carbonate (2.5 g) in anhydrous acetonitrile (20 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (720 mg, 2.00 mmol) in anhydrous acetonitrile (5 mL) was added to the reaction mixture which was maintained 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on silica gel gave the desired product XVIIIc in appreciable yields.

¹H nmr (CD₃OD, TMS): 61.53 (6H, s), 2.36 (3H, s), 4.84 (2H, s), 5.04 (2H, s), 7.46 (1H, s, -triazole-H), 7.68 (1H, s).

The purified derivative XVIIIc (100 mg, 0.35 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 3-aminotriazole pyridoxine XIXc in good yield.

¹H nmr (DMSO-d₆, TMS): 62.32 (3H, s), 4.75 (2H, s), 5.11 (2H, s), 7.35 (1H, s, -triazole-H), 7.58 (1H, s).

Example 9 Synthesis of Imidazole Substituted Pyridoxine Analogue of Formula XXIIa

A mixture of 2-methylimidazole (164 mg, 2.00 mmol) and pulverized anhydrous potassium carbonate (2.5 g) in anhydrous acetonitrile (20 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (720 mg, 2.00 mmol) in anhydrous acetonitrile (5 mL) was added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on silica gel gave the desired product XXIa in appreciable yields.

¹H nmr (CDCl₃, TMS): δ 1.51 (6H, s), 2.40 (3H, s), 2.42 (3H, s, immidazole-CH₃), 4.50 (2H, s), 4.88 (2H, s), 6.65 (1H, s, imidazole-H), 6.93 (1H, s, imidazole-H), 7.82 (1H, s).

The purified derivative XXIa (100 mg, 0.35 mmol) was then dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 2-methylimidazole pyridoxine XXIIa in good yield.

¹H nmr (DMSO-d₆, TMS): δ 2.40 (3H, s), 2.21 (3H, s, imidazole-CH₃), 5.11 (2H, s), 5.20 (2H, s), 6.77 (1H, s, imidazole-H), 6.92 (1H, s, imidazole-H), (2H, s), 7.47 (1H, s).

Example 10 Synthesis of Imidazole Substituted Pyridoxine Analogue of Formula XXIIb

Following Scheme 9, a mixture of 4-methylimidazole (164 mg, 2.00 mmol) and pulverized anhydrous potassium carbonate (2.5 g) in anhydrous acetonitrile (20 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (720 mg, 2.00 mmol) in anhydrous acetonitrile (5 mL) was added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on silica gel gave the desired product XXIb in appreciable yields.

¹H nmr (CDCl₃, TMS): δ 1.50 (6H, s), 2.43 (3H, s), 2.20 (3H, s, immidazole-CH₃), 4.54 (2H, s), 4.92 (2H, s), 6.52 (1H, s, imidazole-H), 7.42 (1H, s, imidazole-H), 7.94 (1H, s).

The purified derivative XXIb (100 mg, 0.35 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 4-methylimidazole pyridoxine XXIIb in good yield.

¹H nmr (CDCl₃, TMS): δ 2.20 (3H, s, imidazole-CH₃), 2.46 (3H, s), 4.72 (2H, s), 4.90 (2H, s), 6.48 (1H, s, imidazole-H), 7.24 (1H, s, imidazole-H), 7.84 (1H, s).

Example 11 Synthesis of Imidazole Substituted Pyridoxine Analogue of Formula XXIIc

Following Scheme 9, a mixture of 4-nitroimidazole (172 mg, 2.00 mmol) and pulverized anhydrous potassium carbonate (2.5 g) in anhydrous acetonitrile (20 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (720 mg, 2.00 mmol) in anhydrous acetonitrile (5 mL) was added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on silica gel gave the desired product XXIc in appreciable yield.

¹H nmr (CDCl₃, TMS): δ 1.52 (6H, s), 2.42 (3H, s), 4.60 (2H, s), 5.09 (2H, s), 7.46 (1H, s, imidazole-H), 7.69 (1H, s, imidazole-H), 8.01 (1H, s).

The purified derivative XXIc (110 mg, 0.35 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 4-nitroimidazole pyridoxine XXIIc in good yield.

¹H nmr (DMSO-d₆, TMS): δ 2.42 (3H, s), 4.74 (2H, s), 5.35 (2H, s), 7.77 (1H, s, imidazole-H), 7.95 (1H, s), 8.14 (1H, s, imidazole-H).

Example 12 Synthesis of Imidazole Substituted Pyridoxine Analogue of Formula XXIId

Following Scheme 9, a mixture of 2-aminoimidazole (132 mg, 2.00 mmol) and pulverized anhydrous potassium carbonate (2.5 g) in anhydrous acetonitrile (20 mL) was stirred at 0° C. for 15 minutes. The bromide VII (q=1) (720 mg, 2.00 mmol) in anhydrous acetonitrile (5 mL) was added to the reaction mixture which was maintained at 0° C. for the next 30 minutes. Routine work-up gave the crude product. Purification of the crude mixture by chromatography on silica gel gave the desired product XXId in appreciable yield.

¹H nmr (CD₃OD, TMS): δ 1.52 (6H, s), 2.35 (3H, s), 4.68 (2H, s), 4.88 (2H, s), 6.44 (1H, s, imidazole-H), 6.53 (1H, s, imidazole-H), 7.64 (1H, s).

The purified derivative XXId (106 mg, 0.35 mmol) was then dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the 2-aminoimidazole pyridoxine XXIId in good yield.

¹H nmr (CD₃OD, TMS): δ 2.15(3H, s), 4.55 (2H, s), 4.75 (2H, s), 6.24 (1H, s, imidazole-H), 6.32 (1H, s, imidazole-H), 7.23 (1H, s).

Example 13 Synthesis of Guanidine Substituted Pyridoxine Analogue of Formula XXV

A solution of 5-aminopyridoxine derivative X (t=1) (90 mg, 0.43 mmol) in anhydrous dichloromethane (5 mL), was added a solution of triflated BOC-guanidine derivative (153.6 mg, 0.39 mmol) in anhydrous dichloromethane (5 mL) and anhydrous triethylamine (60 μL). This solution was stirred at room temperature for one hour. The reaction mixture was washed with 2M sodium bisulfite (10 mL), followed by saturated aqueous NaHCO₃. Evaporation of the dichloromethane left a residue that was purified by chromatography on silica gel to give the guanidine derivative XXIII in appreciable yield.

¹H nmr (CD₂Cl₂, TMS): δ 1.46 (9H, s), 1.48 (9H, s), 1.54 (6H, s), 4.40 (2H, s), 4.88 (2H, s), 7.95 (1H, s), 8.43 (NH, s), 11.48 (NH, s).

The purified derivative XXIII (100 mg, 0.36 mmol) was then dissolved in 20% trifluoroacetic acid in anhydrous dichloromethane (10 mL) and stirred at room temperature for 1 hour. Purification of the reaction mixture gave the two products XXIV and XXV.

¹H nmr of XXIV (MeOD, TMS): δ 1.64 (6H, s), 2.63 (3H, s), 4.55 (2H, s), 5.10 (2H, s), 7.52 (1H, m), 8.13 (1H, s).

¹H nmr of XXV (DMSO-d₆, TMS): δ 2.54 (3H, s), 3.96 (1H, s), 4.54 (2H, d), 4.81 (2H, s), 5.96 (1H, br s, NH), 7.44 (3H, br, NH), 8.07 (1H, s).

Example 14 Synthesis of Aminotriazole Substituted Pyridoxal Analogue of Formula XXVIII

A pyridoxal derivative VIII (r=0) (400 mg, 1.91 mmol) and 3-aminotriazole (178 mg, 2.12 mmol) in anhydrous toluene (20 mL), was heated in a three neck flask, fitted with a condenser and a Dean Stark trap, at 100° C. for 24 hours. Routine workup gave the crude product, which was then purified by chromatography on silica gel to give the protected triazoline derivative XVI in modest yields.

¹H nmr (CD₃OD, TMS): δ 1.58 (6H, s), 2.46 (3H, s), 3.31(1H, s), 5.31 (2H, s), 8.41 (1H, s), 9.23 (1H, s, -triazoline CH).

The fully protected pyridoxine derivative XXVI (206 mg, 0.88 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the triazoline substituted pyridoxine XXVII in good yield.

¹H nmr (CD₃OD, TMS: δ 2.46 (3H, s), 5.10 (2H, m), 6.78 (1H, s, -triazoline CH), 7.92 (1H, s).

Example 15 Synthesis of Imidazoline Substituted Pyridoxine Analogue of Formula XXIX

A pyridoxal derivative VIII (r=0) (2.07 g, 10.00 mmol) and 2-methylimidazoline (1.68 g, 20.00 mmol) in anhydrous toluene (50 mL), was heated in a three neck flask, fitted with a condenser and a Dean Stark trap, at 100° C. for 24 hours. Routine workup gave the crude product, which was then purified by chromatography on silica gel to give the protected imidazoline derivative XXVIII in modest yields.

¹H nmr (CD₃OD, TMS): δ 1.56 (6H, s), 2.38 (3H, s), 3.76 (4H, s), 4.98 (2H, s), 6.63 (1H, s, vinylic CH), 7.26 (1H, s, vinylic-CH), 8.20 (1H, s).

The fully protected pyridoxine derivative XXVIII (100 mg, 0.43 mmol) was dissolved in 80% aqueous acetic acid (5 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the imidazoline XXIX in good yield.

¹H nmr (CD₃OD, TMS): δ 2.14 (3H, s), 3.09 (2H, s), 3.56 (4H, s), 4.74 (1H, s, vinylic CH), 4.87 (1H, s, vinylic-CH), 5.24 (1H, d, NH) 7.26 (1H, s).

Example 16 Synthesis of Imidazole Substituted Pyridoxine Analogue of Formula XXXI

Pyridoxal derivative VIII (r=0) (800 mg, 3.82 mmol) and 2-aminoimidazole (346 mg, 4.2 mmol) in anhydrous dimethyl sulfoxide (75 mL), was heated in a three neck flask, fitted with a condenser and a Dean Stark trap, at 100° C. for three days. Routine workup gave the crude product, which was then purified by chromatography on silica gel to give the protected cyclic guanidine derivative XXX in modest yield.

¹H nmr (MeOD, TMS): δ 1.58 (6H, s) 2.49 (3H, s), 5.26 (2H, s), 7.07 (2H, s imidazole-H), 8.49 (1H, s), 9.27 (1H, s-vinylic H).

The protected pyridoxine derivative XXX (10 mg, 0.44 mmol) was dissolved in 80% aqueous acetic acid (5 mL) and heated at 60° C. for 1 hour. Purification by chromatography on silica gel gave the cyclic guanidine XXXI in good yield.

¹H nmr (MeOD, TMS): δ 2.45 (3H, s), 4.96 (2H, m), 5.12 (2H, dd), 6.43 (1H, d), 7.92 (1H, s).

Example 17 Synthesis of Aminophenylamidine Substituted Pyridoxine Analogue of Formula XXXV

In a 250 mL three-necked flask fitted with a condenser and a Dean stark trap was added protected pyridoxine aldehyde VIII (r=0) (3.31 g, 16 mmol), 4-cyano aniline (1.89 g, 15.9 mmol), p-toluenesulfonic acid (0.3 g, 1.6 mmol) and dry benzene (60 mL). The reaction mixture was then heated to reflux for 16 hours. The reaction mixture was then washed with 2N NaOH (5 mL) followed by brine (10 mL) and the organic layer dried with anhydrous Na₂SO₄. Removal of the solvent gave the crude product, which was purified by silica gel column chromatography, using the eluant 100:1/CH₂Cl₂: 2M NH₃-MeOH. XXXII was obtained as a pure yellow solid. (2.98 g, 61% yield).

¹HNMR (CDCl₃) δ 1.58 (s 6H) 2.49 (s, 3H) 5.27 (s, 2H) 7.20 (d, 2H) 7.68 (d, 2H) 8.31 (s, 1H) 8.44 (s, 1H).

The Schiff base XXXII (358 mg, 1.0 mmol) was dissolved in HOAc (5 mL) and the solution cooled to 0° C. Sodium borohydride (57 mg, 1.5 mmol) was added in portions, while stirring continued for 10 minutes at 0° C., and then at room temperature for another 10 minutes. The reaction was quenched by adding 5 N NaOH (1.8 mL) to bring the pH of the solution to 9. The product was then extracted with diethyl ether (2×10 mL) and dried over anhydrous Na₂SO₄. Evaporation of the solvent, followed by purification on silica gel column chromatography in EtOAc: Hexanes/1:1 gave XXXIII as a pale yellow solid (300 mg, 90%).

¹HNMR (CDCl₃) δ 1.54 (s, 6H), 2.40 (s, 3H) 4.20 (s, 2H) 4.36 (br, 1H) 4.84 (s, 2H) 6.62 (d, 2H) 7.45 (d, 2H) 8.00 (s, 1H).

Into a solution of the amine XXXIII (81 mg, 0.3 mmol) in ethanol (6 mL) at 0° C. was bubbled hydrogen chloride gas (dry) for 30 min. The reaction mixture was slowly allowed to reach the room temperature and then stirred at this temperature for 16 hours. The solution was again cooled to 0° C., and then degassed by bubbling N₂ through it for 2 h. On evaporation of the solvent XXXIV was obtained as a light yellow solid.

¹HNMR (MeOD) δ 1.57 (t, 3H) 2.64 (s, 3H) 4.54 (q, 2H) 4.65 (s, 2H) 5.14 (s, 2H) 6.82 (d, 2H) 7.89 (d, 2H) 8.00 (s, 1H).

In a sealed high pressure flask containing the crude compound XXXIV (106 mg, 0.3 mmol) was added 2 M NH₃-MeOH (10 mL) and the mixture cooled to −78° C., and stirred at this temperature for 15 minutes. The reaction mixture was gradually warmed to rt, and then heated to 80° C. for 2 h. The reaction mixture was again cooled to −78° C. and then the sealed flask was opened. The solution was then transferred to a round-bottomed flask and the solvent evaporated to dryness. Purification on silica gel column using 100:20:1/CH₂Cl₂: MeOH: H₂O as eluent gave XXXV as white solid (88 mg, 82%).

¹HNMR (MeOD) δ 2.43 (s, 3H) 4.45 (s, 2H) 4.96 (s, 2H) 6.78 (d, 2H) 7.64 (d, 2H) 7.84 (s, 1H).

Example 18 Synthesis of Aminophenylamidine Substituted Pyridoxine Analogue of Formula XL

The procedure for the preparation of XXXVI was similar to that used for the synthesis of compound XXXIIII. The crude product, XXXVI, was an auburn solid and was used in the next step without purification.

¹HNMR (CDCl₃) δ 1.59 (s, 6H) 2.49 (s, 3H) 5.30 (s, 2H) 7.06 (d, 2H) 7.51 (d, 2H) 8.30 (s, 1H) 8.47 (s, 1H).

The procedure for the preparation of XXXVII was similar to that used for the synthesis of compound XXXIII. The crude product, XXXVII, was purified by silica gel column, using 5:1:1.5/CH₂Cl₂:MeOH:H₂O, as eluant to give a yield white solid. (66% yield).

¹HNMR (CDCl₃) δ 1.55 (s, 6H) 2.41 (s, 3H) 3.68 (br, 1H) 4.12 (d, 2H) 4.87 (s, 2H) 6.51 (d, 2H) 7.27 (d, 2H) 8.01 (s, 1H).

Compound XXXVII (665 mg, 1.83 mmol) was dissolved in diglyme (15 mL), followed by the addition of Pd (PPh₃)₄ (63 mg, 0.05 mmol). The mixture was stirred for 10 min, and then p-cyanophenylboronic acid (269 mg, 2.01 mmol) was added to the reaction mixture followed by sodium bicarbonate (461 mg in 8 mL H₂O, 5.49 mmol). The reaction mixture was heated to 95° C. in an oil bath for 5 min, and then stirred at room temperature for 1.5 h. On evaporation of the solvent a dark purple crude product, XXXVIII, was obtained which was purified by silica gel column chromatography (1:1/EtOAc: Hexane, 32% yield.).

¹H NMR (CDCl₃) δ 1.56 (s, 6H) 2.42 (s, 3H) 3.96 (br, 1H) 4.22 (d, 2H) 4.91 (s, 2H) 6.72 (d, 2H) 7.47 (d, 2H) 7.65 (q, 4H) 8.06 (s, 1H).

The syntheses of compound XXXIX and XL were accomplished by the procedures outlined for compounds XXXIV and XXXV. Overall yield of XL was 63% for the two steps.

¹HNMR (XXXIX) (MeOD) δ 1.62 (t, 3H) 2.62 (s, 3H) 4.56 (s, 2H) 4.63 (q, 2H) 5.17 (s, 2H) 6.78 (d, 2H) 7.60 (d, 2H) 7.83 (d, 2H) 8.02 (s, 1H) 8.05 (d, 2H).

¹HNMR (XL) (MeOD) δ 2.43 (s, 3H) 4.39 (s, 2H) 4.98 (s, 2H) 6.76 (d, 2H) 7.54 (d, 2H) 7.80 (m, 4H) 7.89 (s, 1H).

Example 19 Synthesis of Arylurea and Arylthiourea Substituted Pyridoxine Analogues of Formulas XLIII and XLIV

The azide IX (t=1) (790 mg, 3.4 mmol) was dissolved in 80% aqueous HOAc (40 mL) and heated at 60° C. for 16 hours, to hydrolyze the isopropylidene group. Co-distillation with toluene to remove acetic acid gave the crude product, which was purified by silica gel column chromatography. (1:1→4:1/EtOAc: Hexane), giving XLI as a white solid (410 mg, 63% yield.)

¹H NMR (MeOD) δ 2.43 (s, 3H) 4.42 (s, 2H) 4.92 (s, 2H) 7.86 (s, 1H).

To the de-blocked azide XLI (388 mg, 2.0 mmol) in dry DMF (10 mL) was added imidazole (545 mg, 8.8 mmol) and TBDPSiCl (1.2 ml, 4.4 mmol). The reaction mixture was then heated to 50° C. and kept stirring for overnight. The reaction was then cooled to room temperature, and then extracted with diethyl ether (2×25 mL), followed by washing the ether extract by water (2×10 mL) and brine (1×10 mL). The ethereal solution was then dried over MgSO₄ and solvent evaporated to dryness to give XLII as a white solid. (1.3 g, 97% yield).

¹H NMR (CDCl₃) δ 0.87 (s, 9H) 0.96 (s, 9H) 2.15 (s, 3H) 4.49 (s, 2H) 4.52 (s, 2H) 7.38 (m, 20H) 8.03 (s, 1H).

To the compound XLII (1.3 g, 1.94 mmol) dissolved in methanol (20 mL) was added Lindlar catalyst (600 mg) and the mixture hydrogenated under a stream H₂ for 1.5 hours to complete the reaction. The routine work up and purification gave the amine XLIII (0.95 g, 76%).

¹H NMR (CDCl₃) δ 0.83 (s, 9H) 0.96 (s, 9H) 1.51 (br, 2H) 2.11 (s, 3H) 3.89 (s, 2H) 4.57 (s, 2H) 7.33 (m, 20) 8.09 (s, 1H).

A mixture of XLIII (645 mg, 1.0 mmol) and the 4-fluorophenylisocyanate (0.12 mL, 1.0 mmol) in dry toluene (10 mL) was refluxed for 16 hours. After the removal of the solvent, the crude product was purified by silica gel column chromatography, using 100:1→100:2/CH₂Cl₂: 95% EtOH as eluent, giving XLIV a white solid (619 mg, 79%).

¹H NMR (CDCl₃) δ 0.87 (s, 9H) 0.97 (s, 9H) 2.13 (s, 3H) 4.87 (d, 2H) 4.62 (s, 2H) 5.05 (br, 1H) 6.15 (br, 1H) 7.33 (m, 20H) 8.19 (s, 1H). ¹⁹F NMR CDCl₃ δ-120.40 (s).

To a solution of XLIV (391 mg, 0.5 mmol) in dry THF (10 ml) was added TBAF (0.32 ml, 2.2 mmol) and stirred at room temperature for 16 hours. Removal of the solvent followed by purification by silica gel column chromatography using 20:1→10:1/CH₂Cl₂: MeOH, as eluent gave XLV as a white solid. (135 mg, 89%).

¹H NMR (MeOD) δ 2.40 (s, 3H) 4.39 (s, 2H) 4.94 (s, 2H) 6.98 (m, 2H) 7.34 (m, 2H) 8.78 (s, 1H). ¹⁹F NMR (MeOD) 6-123.51 (m).

The syntheses of compounds XLVI and XLVII followed the procedure outlined for XLIV and XLV using 4-fluorophenylthioisocyanate in place of 4-fluorophenylisocyanate. Yields obtained were 65% for XLVI and 60% for XLVII.

¹H NMR (XLVI) (CDCl₃) δ 0.82 (s, 9H) 0.88 (s, 9H) 2.09 (s, 3H) 4.52 (s, 2H) 5.00 (d, 2H) 5.29 (s, 1H) 6.36 (t, 1H) 7.33 (m, 20H) 8.02 (s, 1H). ¹⁹F NMR (CDCl₃) 6-113.42 (s).

¹H NMR (XLVII) (MeOD) δ 2.41 (s, 3H) 3.07 (m, 1H) 3.54 (m, 1H) 4.91 (s, 2H) 7.09 (t, 2H) 7.32 (q, 2H) 7.89 (s, 1H). ¹⁹F NMR (MeOD) 6-118.80 (m).

Example 20 In Vivo Assay—Coronary Artery Ligation

Myocardial infarction was produced in male Sprague-Dawley rats (300-400 g) by occlusion of the left coronary artery. The rats were housed in clear cages at room temperature and humidity on a 12 hour light-dark cycle. Food and water were supplied ad libitum. Rats were anaesthized and the skin was incised along the left sternal border and the fourth rib was cut proximal to the sternum and a retractor was inserted. The pericardial sac was opened and the heart externalized. The left anterior descending coronary artery was ligated approximately 2 mm from its origin on the aorta using a 6-0 silk suture. The heart was then repositioned in the chest and the incision closed via purse-string sutures. Sham-operated rats underwent substantially identical treatment except that the artery was not ligated. All animals were allowed to recover, receiving food and water ad libitum for 21 days. Hemodynamic and histological assessments were made.

Occlusion of the coronary artery in rats has been shown to produce myocardial cell damage, which results in scar formation in the left ventricle and heart dysfunction. While the complete healing of the scar typically occurs within 3 weeks of the coronary occlusion, mild, moderate and severe stages of congestive heart failure have been reported to occur at 4, 8 and 16 weeks after ligation. Accordingly, the contractile dysfunction seen at 3 weeks after coronary occlusion in rats is due to acute ischemic changes.

Rats were divided at random into five groups: sham operated, coronary artery ligated (untreated), coronary artery ligated (pyridoxal-5′-phosphate (PLP) treatment), and coronary artery ligated (compound XXIIb treated). Treatment with PLP and compound XXIIb began 1 hour after coronary occlusion (or sham operation), and continued for 21 days. PLP or compound XXIIb (10 mg/kg) were administered daily (9 AM) by gastric tube. This dosage was chosen based on previous experience with PLP.

Mortality in all groups occurred only within the first 24 h after coronary ligation. While in the untreated group 50% of the rats died, the mortality rate was significantly reduced in the treated groups.

Example 21 In Vivo—Hemodynamic Changes

The animals prepared as described in Example 20 were anaesthetized on the 21^(st) day with an injected cocktail of ketamine hydrochloirde and xylazine. To maintain adequate ventilation, the trachea was intubated. The right carotid artery was exposed and a microtip pressure transducer was introduced (Model SPR-249, Millar, Houston, Tex.) into the left ventricle. The catheter was secured with a silk ligature around the artery, and various hemodynamic parameters including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), rate of contraction (+dP/dt), and rate of relaxation (−dP/dt), were recorded and calculated with Acknowledge 3.1 software (Biopac Systems Inc.). The animals were allowed 5-10 minutes to stabilize, after which parameters were measured as averages over three readings.

Average +dP/dt and −dP/dt values were reduced in the untreated group compared to the sham control group. The experimental groups receiving P5P or compound XXIIb experienced recoveries in +dP/dt (rate of contraction) and −dP/dt (rate of relaxation) values.

LVSP (left ventricular systolic pressure) was decreased in the untreated group compared to the sham control group, after 21 days of coronary ligation. Average LVEDP was increased in the untreated group, compared to the sham control group. Treatment with PLP, or compound XXIIb yielded similar reduced rises in LVEDP in response to coronary occlusion.

Example 22 In Vivo—Infarct Size and Scar Mass

After 21 days, once the hemodynamic data were obtained, the animals were sacrificed and both average dry scar mass to left ventricle mass (n=5/group) and infarct size (n=5/group) were measured. For infarct size measurement, hearts from the untreated and PLP/compound XXIIb-treated groups were fixed in formalin and embedded in paraffin. Six evenly spaced slices were cut across the left ventricle. 5 μM sections were cut from each slice and mounted. Sections were stained with Trichrome to discriminate between fibrous scar and noninfarcted tissue. Using the free-drawing line tool in Scion Image v4.02b, infarct internal perimeter and left ventricle internal perimeter lengths were traced per oculum for each section. Noteworthy transmural scars were taken into account. Infarct size was then expressed as the average scar perimeter/ventricle perimeter ratio. Scar mass measurements were obtained by drying excised scar tissue and left ventricles at 50-60° C. for 72 hours.

The PLP-treated group had a reduced dry scar to left ventricle mass ratio. The compound XXIIb-treated group had scar/ventricle ratio reductions similar in magnitude to that seen in the PLP group. In the untreated control group, average infarct size (as a percentage of left ventricle size) was about 45%; in the PLP and compound XXIIb groups, the infarct size was reduced.

Example 23 In Vivo—Hypertrophy

Hypertrophy is a physiological condition of enlargement (increased mass) due to increased stress. Cardiac hypertrophy is assessed by calculating the heart to body mass ratio. Treatment with PLP or compound XXIIb results in a decrease in cardiac hypertrophy, in the rat model described in Example 20.

Example 24 Inhibition of Platelet Aggregation

Platelet rich plasma (PRP) was obtained by drawing whole blood into sodium citrate tubes (3.2%) and centrifuging at 700 rpm for 10 minutes. Platelet poor plasma (PPP) was obtained by centrifuging the remainder of the sample util the platelets were removed (3200 rpm for 10 minutes). The PRP was adjusted to a count of 280×10⁹/L using a mixture of PRP and PPP. The incubation mixture consisted of 200 μL of platelets and 25 μL of the appropriate compound (18 mM stock for a final concentration of 2 mM and 4.5 mM stock for a final concentration of 500 μM), rendering an approximate final platelet count in the incubation mixture of 250×10⁹ /L. After incubation (30 minutes at room temperature, the cuvettes were incubated for 3 minutes at 37° C., transferred to the mixing wells and after the baselines transmittances were measured in an aggregometer (Chrono-Log 4), 25 μL of agonist was added to give a final concentration of 4 μM ADP, 1 μg/mL collagen, 5 μg/mL collagen or 12 μM thrombin receptor activating peptide (TRAP). Final transmittances were then measured in the aggregometer (lower percent transmittance represents higher aggregation). Agonists concentrations were chosen based on previous experience that indicated that these were the smallest concentrations that would give the full extent of aggregation in the normal population. Table 24-1 gives the results of the extent of aggregation for several compounds as percentage amplitude read directly from the aggregometer. TABLE 24-1 Extent of aggregation as a percentage amplitude, read directly from the aggregometer. Compound 5 μg/mL 1 μg/mL 4 μM 12 μM Diluent of Tested collagen collagen ADP TRAP Compound Saline 82% 87% 83% 93% Control DMSO 85% 86% 55% 88% Control XL 5% 4% 4% 4% saline  2 Mm XL 52% 4% 9% 3% saline 500 μM XLVII 49% 30% 31% 42% 20% DMSO  2 mM in saline XXXV 67% 79% 15% 11% saline  2 mM XXXV 75% 83% 70% 90% saline 500 μM XXIIb 77% 32% 46% 84% 1 part  2 mM DMSO 1 part saline XXIIb 82% 79% 59% 91% 1 part 500 μM DMSO 1 part saline PLP 89% 84% 16% 91% saline  2 mM PLP 87% 89% 69% 90% saline 500 μM

Example 25 In Vivo—Neuroprotection in Rat Embolic Stroke Model

Compound XL (see example 18, scheme 12) was assayed in a rat embolic stroke model.

Male Wistar rats, weighting 300-350 g, were purchased from Charles River, St. Constant, Canada. Embolic focal cerebral ischemia was induced by embolizing a pre-formed clot into the middle cerebral artery (MCA). (For details of the procedure see Wang C X, Yang Y, Shuaib A. A focal embolic model of cerebral ischemia in rats. Brain Res. Protoc. 7:115-120, 2001. Wang C, Yang T and Shuaib A: An improved version of embolic model of brain ischemic injury in the rat. J. Neurosci. Meth. 109:147-151, 2001).

The study consisted of four groups (n=10). Animals were randomly assigned to one of the four groups. Treatments were started at 1 h after MCA occlusion. XL was dissolved in 0.5% DMSO (1.5 ml) and infused over a period of 60 min i.v. In the experimental groups, XL, 20, 40 or 80 mg/kg was infused. In the control group, 0.5% DMSO (1.5 ml) was infused.

The procedures for assessment of infarct volume were detailed previously (Bederson J B, Pitts L H, Tsuji M, Nishimura M C, Davis R L, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472-476, 1986. Yang, Y., Shuaib, A., and Li, Q. Quantification of infarct size on focal cerebral ischemia model of rats using a simple and economical method. J. Neurosci. Meth., 84:9-16, 1998). Briefly, at 48 h after ischemia the anesthetized animal was sacrificed by decapitation and the brain removed. The volume of infarction was measured in the brain sections stained with 2,3,5-triphenyltetrazolium chloride (TTC) solution with following formula, corrected infarct size=the volume of the left hemisphere−(the volume of the right hemisphere-measured infarct volume). The percentage of infarct volume was obtained by calculating the portion of the corrected infarct volume and dividing it by the total volume of the left hemisphere. Brain swelling was determined using a formula: swelling (edema)=(volume of the right hemisphere−volume of the left hemisphere)/volume of the left hemisphere. Hemorrhage transformation was identified from the TTC stain brain sections.

Neurological deficits and seizure activities were recorded at 2, 24 and 48 hours after embolization. Neurological deficits were determined using a modified Bederson's scoring system (Bederson J B, Pitts L H, Tsuji M, Nishimura M C, Davis R L, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472-476, 1986). 0: no observable deficit; 1: forelimb flexion; 2: forelimb flexion plus decreased resistance to lateral push; 3: unidirectional circling; 4: unidirectional circling plus decreased level of consciousness. Seizure activity, hemorrhage and mortality were also recorded. Seizure was also recorded. Results are shown in Table 25-1.

The differences of infarction volume were analyzed with one way ANOVA followed by Dunnett's test. Neurological deficit scores were reported as medians and interquartile ranges, considering 25-75^(th) percentile. The neurological scores were analyzed with Kruskal-Wallis test when more than two groups, and analyzed with Mann-Whitney test when comparison was done in two groups. The rates of mortality and seizure occurrence following different treatments were compared with Chi-square test. Differences were considered significant when p<0.05.

Infarction volume and brain edema. Embolizing a pre-formed thrombus resulted in an infarction in the territory irrigated by MCA, mainly located in the cerebral cortex and striatum. In the control group, the infarction volume was 40±2.6% (mean±SEM) at 48 h after the embolization. In the groups administrated XL 10, 20 or 40 mg/kg at 1 h after embolization, the infarction volumes were 30.5±4.8, 26.5±4.4 and 25.9±3.4% respectively, which was significantly different from the control group. Ischemic brain edema was 5.7±1.3, 6.8±1.9, 5.9±1.3 and 7.9±2.1 respectively. There was no significant difference among the groups.

Neurological deficits. Neurological deficits are shown in Table 25-1. In all four groups, the Bederson' score was 3 at 2 after the ischemic injury. Kruskal-Wallis test analysis showed that there were significant differences among the groups at 48 h but not at 24 h. Compared to the score recorded at 2 h after the injury, it did not significantly improve at either 24 h or 48 h after the injury in the control group. In the rats receiveing the drug injection of 10, 20 or 40 mg/kg at 2 h after the injury, neurological scores improved significantly at 24 h after the injury when compared with that at 2 h after the injury (P<0.05). Neurological scores also improved significantly at 48 h after the injury when compared with that at 2 h after the injury (P<0.05).

Seizure occurrence. Seizure occurred in 2 rats in the control group, 3 rats in the 10 mg/kg group, 2 rats in the 20 mg/kg group and 2 rats in the 40 mg/kg group.

Hemorrhage rate. Hemorrhage was observed in 1 rats in the control group, 2 rats in the 10 mg/kg group, 1 rat in the 20 mg/kg group and 2 rats in the 40 mg/kg group.

Mortality rate. Pre-maturely death occurred 1 rat in control group, 1 in the 10 mg/kg group and 1 in the 20 mg/kg group. TABLE 25-1 Neurological deficits in rats received different doses of XL after injury^(a) Saline 10 mg^(b) 20 mg 40 mg Group n = 10 n = 10 n = 10 n = 10  2 h 3 (3—3) 3 (3—3) 3 (3—3)   3 (3—3) 24 h 3 (3—3) 2 (2-3)* 2 (2-3)*   2 (2-3)* 48 h 3 (3—3) 2 (2-3)* 2 (2-3)* 2.5 (2—2)* ^(a)The neurological deficits were recorded at 2 and 24 h after the ischemic injury. The neurological deficits scores are expressed as median and interquartile ranges, the 25-75^(th) percentile are shown in the parenthesis ^(b)The drugs were administered at 1 h after the ischemic injury. *denotes significant difference from the value at 2 h after the ischemic injury.

Example 26 In Vivo—Assay in Rat Cardiac Ischemia Reperfusion Model

Compound XL (see example 18, scheme 12) was assayed in a rat cardiac ischemia reperfusion model

Seventy-nine male Wistar rats (200-300 g, Tuck, Rayleigh, Essex, U.K.) were anesthetized with thiopentone sodium (Intraval®, 120 mg/kg i.p.; Rhone-Merrieux, Essex, U.K.). The rats were tracheotomized, intubated and ventilated with a Harvard ventilator (70 strokes/min, tidal volume: 8-10 ml/kg, inspiratory oxygen concentration: 30%). Body temperature was maintained at 38±1° C. The right carotid artery was cannulated and connected to a pressure transducer (Spectramed, P23XL) to monitor mean arterial blood pressure (MAP) and heart rate (HR), which were continuously recorded on a 4-channel Grass 7D polygraph recorder (Grass, Mass., U.S.A.). The right jugular vein was cannulated for the administration of drugs. Subsequently, a lateral thoracotomy was performed and the heart was suspended in a temporary pericardial cradle. A snare occluder was placed around the left anterior descending coronary artery (LAD). After completion of the surgical procedure the animals were allowed to stabilize for 30 min before LAD ligation. The coronary artery was occluded at time 0 by tightening of the occluder. After 25 min of acute myocardial ischemia, the occluder was re-opened to allow the reperfusion for 2 h. Following the 2 h reperfusion period, the coronary artery was re-occluded and Evans Blue dye (1 ml of 2% w/v) was injected into the left ventricle, via the right carotid artery cannula, to distinguish between perfused and non-perfused area at risk (AR) sections of the heart. The Evans Blue solution stains the perfused myocardium, while the occluded vascular bed remains uncolored. The animals were killed with an overdose of anesthetic and the heart excised. It was sectioned into slices of 3-4 mm, the right ventricular wall was removed, and the AR (pink) was separated from the non-ischemic (blue) area. The AR was cut into small pieces and incubated with p-nitroblue tetrazolium (NBT, 0.5 mg/ml) for 40 min at 37° C. In the presence of intact dehydrogenase enzyme systems (viable myocardium), nitro-blue tetrazolium (NBT) forms a dark blue formazan, whilst areas of necrosis lack dehydrogenase activity and therefore fail to stain. Pieces were separated according to staining and weighed to determine the infarct size as a percentage of the weight of the AR.

The following experimental groups were studied:

-   -   (1) Sham-operation (no occlusion of the LAD) plus administration         of saline (Sham-Saline, 1 ml/kg i.v. n=9).         -   (2) LAD-occlusion (25 min) and reperfusion (2 h) plus             administration of saline (myocardial infarction             (“MI”)-Saline; 1 ml/kg i.v. bolus, 5 min prior to LAD             occlusion) (n=10).     -   (3) LAD-occlusion and reperfusion plus administration of XL (25         mg/kg i.v. bolus at 5 min prior to LAD occlusion) (n=10).

All data were expressed as mean±s.e.mean of n observations, where n represented the number of animals studied. Infarct size was analyzed by 1-factorial ANOVA, followed by a Bonferroni's post hoc test for multiple comparisons. Hemodynamic data were analyzed by 2-way ANOVA followed by a Bonferroni's test. A P-value of less than 0.05 was considered to be statistically significant (when compared to MI-Saline).

The baseline values of mean arterial pressure (MAP) in all groups of animals were not significantly different between groups (P>0.05, Table 26-1). The MAP of rats subjected to LAD occlusion was significantly lower at 15 min of regional myocardial ischemia (P<0.05, when compared to sham operated animals, Table 26-1). In addition there was a progressive decline in MAP during the reperfusion period (P<0.05, Table 26-1). The fall in MAP observed in MI-rats treated with the test compound XL (25 mg/kg i.v.)] was similar to the fall in MAP observed in the rats subjected to myocardial ischemia and treated with Saline. (P>0.05, Table 26-1).

Baseline values of heart rate (HR) in all groups of animals were not significantly different between groups (P>0.05, Table 26-1). In rats subjected to myocardial ischemia and reperfusion, there was no change in HR. The test compound had no significant effect on HR in rats subjected to regional myocardial ischemia and reperfusion (P>0.05, Table 26-1).

Baseline values of the power ratio index (PRI) in all groups of animals were not significantly different between groups (P>0.05, Table 26-1). In rats subjected to myocardial ischemia and reperfusion, there was a progressive fall in PRI (P>0.05, Table 26-1). The fall in PRI observed in MI-rats treated with the test compound XL (25 mg/kg i.v.)] was similar to the fall in PRI observed in the MI-rats treated with Saline. (P>0.05, Table 26-1).

The mean values for the area at risk of infarction (AR) ranged from 46±3% to 58±3% and were similar in all animal groups studied (P>0.05, FIG. 1 a). In rats, which were treated with saline (vehicle), occlusion of the LAD (for 25 min) followed by reperfusion (for 2 h) resulted in an infarct size of 48±3% of the AR (MI-Saline, n=10).

Intravenous administration of XL (25 mg/kg i.v. bolus, 5 minutes before the onset of regional ischemia) resulted in a significant reduction in infarct size (P<0.05, FIG. 1 b).

Thus intravenous administration of the test compound XL results in a significant reduction in the infarct size caused by regional myocardial ischemia and reperfusion in an anesthetized mammalian patient. TABLE 26-1 Table illustrating the alterations in hemodynamic parameters throughout the experiment; 25 mins LAD occlusion, 2 h reperfusion Occlusion (mins) Reperfusion (mins) n Baseline 15 25 60 120 Sham Saline 9 MAP 147 ± 5 149 ± 9* 146 ± 9 133 ± 9* 122 ± 5* HR 421 ± 10 424 ± 13 423 ± 14 410 ± 11 403 ± 11 PRI  62 ± 2  36 ± 4  61 ± 4  55 ± 4  48 ± 2 MI Saline 10 MAP 134 ± 6 119 ± 9 117 ± 9  87 ± 9  91 ± 6 1 ml/kg saline HR 427 ± 11 427 ± 13 423 ± 10 401 ± 12 397 ± 9 t = −5 mins PRI  57 ± 3  52 ± 5  51 ± 4  35 ± 4  36 ± 2 XL 8 MAP 121 ± 7 119 ± 6 118 ± 6  89 ± 8  70 ± 7 25 mg/kg i.v; B HR 443 ± 9 435 ± 11 460 ± 10 412 ± 10 414 ± 13 t = −5 mins PRI  54 ± 3  52 ± 3  55 ± 3  36 ± 3  29 ± 3 n = number of animals per group; t = time before occlusion (t = 0); MAP = mean arterial pressure (mmHg); HR = heart rate (beats per min); PRI = pressure rate index (mmHg/min 10⁻³); B = bolus injection; I = infusion; oral administration was via a gavage. *Significant statistical difference when compared with MI Saline (P < 0.05).

Example 27 Inhibition of Platelet Aggregation

XL was capable of inhibiting the aggregation of platelets drawn from patients known to have heparin induced thrombocytopenia (HIT). Platelets from HIT patients were challenged with 0.5 U/ml concentration of heparin in the presence and absence of drug. At 500 micromolar XL was able to completely suppress the aggregation. XL is useful as an antiplatelet treatment for HIT patients who cannot be administered heparin due to the immune reaction with heparin that leads to aggregation rather than thrombolysis.

Example 28 Treatment of Heparin Induced Thrombocytopenia (HIT)

XL inhibited the effects of heparin induced thrombocytopenia (HIT). As shown in Table 28-1, 500 micromolar XL was able to completely suppress the affects of HIT. TABLE 28-1 Amplitude of ATP release Ratio of spike (low Low/High Aggregation Concentration Heparin) Heparin (Low Heparin) Patient 1 0 363 42.4 Present 500 μM 14 1.9 Absent 2 mM 28 2.7 Absent Patient 2 0 286 37.6 Present 500 μM 11 1.6 Absent 2 mM 12 1.6 Absent

Platelets were drawn from HIT patients and challenged with heparin to a final concentration of 0.5 U/ml of low molecular weight heparin, in the presence and absence of the drug. ATP is released and aggregation occurs when patients exhibit HIT. ATP was detected in a lumi aggregometer (Chronolog Corp). A luciferin-luciferase mixture was used to detect ATP release. The luciferin-luciferase mixture was previously calibrated to a known amount of ATP. Thus a spike in ATP release indicated platelet aggregation. As shown by the data, HIT patents have a large amplitude release when there is no XL compound present. When XL compound is present it inhibits this characteristic release of ATP.

Low heparin is low molecular weight heparin and high heparin is unfractionated heparin. The presence of low molecular weight heparin will cause platelet aggregation. All samples were treated with a constant final concentration of heparin; 0.5 U/ml-low and 100 U/ml high. HIT causes platelet aggregation in the Low Concentration of Heparin only, as well as a ration of >5.0 when comparing the Low/High concentration of Heparin. Thus, the data presented in Table 28-1 shows that XL is effective as an treatment for HIT patients whom otherwise cannot be administered heparin due to the immune reaction with heparin that leads to aggregation rather than thrombolysis.

It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds.

Although embodiments of the invention have been described above, it is not limited thereto, and it will be apparent to persons skilled in the art that numerous modifications and variations form part of the present invention insofar as they do not depart from the spirit, nature, and scope of the claimed and described invention. 

1. A method of inhibiting ischemia in a mammalian patient comprising administering a therapeutically effective amount of a compound of Formula V:

or a pharmaceutically acceptable acid addition salt thereof, wherein: R₅ is CH₂OH or CHO; R₁ is

n is an integer of 1 to 5; R₂, R₃, and R₄ are each independently hydrogen; alkyl; aryl or biaryl, wherein the aryl or biaryl can be substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy; amino; acylamino; anilino, wherein the aniline ring can be substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy; nitro; or guanidino.
 2. The method of claim 1 wherein said compound is administered to induce a plasma level of said compound of between about 100 nM and 10,000 μM.
 3. The method of claim 2 wherein said plasma level is between 200 μM and 4000 μM.
 4. The method of claim 3 wherein said plasma level is between 500 μM and 2000 μM.
 5. The method of claim 1 wherein said therapeutically effective dose is between about 0.1 to 100 mg/kg body weight per day.
 6. The method of claim 5 wherein said compound is administered intravenously, orally, sublingually, intraperitoneally, transdermally, intramuscularly or subcutaneously.
 7. The method of claim 1 wherein said compound is administered intravenously.
 8. The method of claim 1 wherein said compound is administered orally.
 9. The method of claim 1 wherein the compound is


10. The method of claim 3 wherein the compound is


11. A method of inhibiting platelet aggregation in a mammalian patient comprising administering a therapeutically effective amount of a compound of Formula V:

or a pharmaceutically acceptable acid addition salt thereof, wherein: R₅ is CH₂OH or CHO; R₁ is

n is an integer of 1 to 5; R₂, R₃, and R₄ are each independently hydrogen; alkyl; aryl or biaryl, wherein the aryl or biaryl can be substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy; amino; acylamino; anilino, wherein the aniline ring can be substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy; nitro; or guanidino.
 12. The method of claim 11 wherein said compound is administered to induce a plasma level of said compound of between about 100 nM and 10,000 μM.
 13. The method of claim 11 wherein said therapeutically effective dose is between about 0.1 to 100 mg/kg body weight per day.
 14. The method of claim 13 wherein said dosage is between 1 mg/kg and 100 mg/kg body weight.
 15. The method of claim 14 wherein said dosage is between 5 mg/kg and 80 mg/kg body weight.
 16. The method of claim 13 wherein said compound is administered intravenously, orally, sublingually, intraperitoneally, transdermally, intramuscularly or subcutaneously.
 17. The method of claim 11 wherein said compound is administered intravenously.
 18. The method of claim 11 wherein said compound is administered orally.
 19. The method of claim 11 wherein the compound is


20. The method of claim 13 wherein the compound is


21. A method of inhibiting cardiovascular or related disease in a mammalian patient comprising administering a therapeutically effective amount of a compound of Formula V:

or a pharmaceutically acceptable acid addition salt thereof, wherein: R₅ is CH₂OH or CHO; R₁ is

n is an integer of 1 to 5; R₂, R₃, and R₄ are each independently hydrogen; alkyl; aryl or biaryl, wherein the aryl or biaryl can be substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy; amino; acylamino; anilino, wherein the aniline ring can be substituted with a cyano, alkyl, alkoxy, amino, hydroxy, halo, nitro, or alkanoyloxy; nitro; or guanidino.
 22. The method of claim 21 wherein said compound is administered to induce a plasma level of said compound of between about 100 nM and 10,000 μM.
 23. The method of claim 21 wherein said therapeutically effective dose is between about 0.1 to 100 mg/kg body weight per day.
 24. The method of claim 23 wherein said dosage is between 1 mg/kg and 100 mg/kg body weight.
 25. The method of claim 24 wherein said dosage is between 5 mg/kg and 80 mg/kg body weight.
 26. The method of claim 23 wherein said compound is administered intravenously, orally, sublingually, intraperitoneally, transdermally, intramuscularly or subcutaneously.
 27. The method of claim 21 wherein said compound is administered intravenously.
 28. The method of claim 21 wherein said compound is administered orally.
 29. The method of claim 21 wherein the compound is


30. The method of claim 23 wherein the compound is


31. The method of claim 21, wherein the cardiovascular or related disease is selected from the group consisting of cerebral ischemia, cerebral hemorrhage, ischemic stroke, and hemorrhagic stroke.
 32. The method of claim 21, wherein the cardiovascular or related disease is hypertension.
 33. The method of claim 32, further comprising administering concurrently a thereapeutic cardiovascular compound selected from the group consisting of an angiotensin converting enzyme inhibitor, a calcium channel blocker, a beta-adrenergic receptor antagonist, a vasodilator, a diuretic, an alpha-adrenergic receptor antagonist, and a mixture thereof.
 34. The method of claim 21, wherein the cardiovascular or related disease is myocardial infarction.
 35. The method of claim 34, further comprising administering concurrently a therapeutic cardiovascular compound selected from the group consisting of an angiotensin converting enzyme inhibitor, a calcium channel blocker, an antithromblytic agent, a beta-adrenergic receptor antagonist, a diuretic, an alpha-adrenergic receptor antagonist, and a mixture thereof.
 36. The method of claim 21, wherein the cardiovascular or related disease is ischemia reperfusion injury.
 37. The method of claim 36, further comprising administering concurrently a therapeutic cardiovascular compound selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, and a mixture thereof.
 38. The method of claim 21, wherein the cardiovascular or related disease is myocardial ischemia.
 39. The method of claim 38, further comprising administering concurrently a therapeutic cardiovascular compound selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, an antithrombolytic agent, a beta-adrenergic receptor antagonist, a diuretic, an alpha-adrenergic receptor antagonist, and a mixture thereof.
 40. The method of claim 21, wherein the cardiovascular or related disease is congestive heart failure.
 41. The method of claim 40, further comprising administering concurrently with a therapeutic cardiovascular compound selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, a vasodilator, a diuretic, and a mixture thereof.
 42. The method of claim 21, wherein the cardiovascular or related disease is arrhythmia.
 43. The method of claim 42, further comprising administering concurrently a therapeutic cardiovascular compound selected from the group consisting of a calcium channel blocker, a beta-adrenergic receptor antagonist, and a mixture thereof.
 44. The method of claim 21, wherein the cardiovascular or related disease is a blood coagulation disorder.
 45. The method of claim 44, further comprising administrating concurrently an antithrombolytic agent.
 46. The method of claim 44, wherein the cardiovascular or related disease is cardiac hypertrophy.
 47. The method of claim 46, further comprising administering concurrently a therapeutic cardiovascular compound selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, and a mixture thereof.
 48. The method of claim 21, wherein the cardiovascular or related disease arises from thrombotic and prothrombotic states in which the coagulation cascade is activated.
 49. The method of claim 48, wherein the disease is deep vein thrombosis, disseminated intravascular coagulopathy, or pulmonary embolism. 